Process for the diagnostic assessment and monitoring, as well as medicaments for the therapy of states of shock in humans

ABSTRACT

Patients suffering from shock illness exhibit increased levels of cyclophilins. Accordingly, methods for diagnosing, monitoring and treating shock patients are disclosed. Medicaments for therapeutically treating patients suffering from shock illness are also provided, for example, Cyclosporin A.

[0001] The subject of the present invention are a new process for the diagnostic assessment and monitoring, as well as a medicament for the therapy of states of shock in humans.

[0002] In the western industrial nations, every year thousands die directly from various forms of shock or the immediate results of the shock. As shock, one understands an acutely occurring faulty regulation of the blood supply of vital organs due to blood pressure decrease. From this acutely occurring faulty regulation, there frequently develop diffuse or selective organ damages and a protracted organ failure which, in about 10 to 30% of the cases, leads to death (cf. e.g. Pschyrembel, Klin. Wörterbuch, de Gruyter Berlin, 1992). Such shock reactions can occur on the basis of acute and chronic infections, in the case of pre-damaged patients (post-operative), treatment with cytostatics etc. or also without recognisable cause, mostly as a result of otherwise harmless bacterial infections. One differentiates between anaphylactic, bacterial, septic, post-traumatic, cardiogenic, haemorrhagic, hypovolaemic, neurogenic and toxic forms of shock. A common factor appears to exist in the coexistence of an altered defence behaviour of the body with regard to frequently harmless microbes on the basis of simultaneous and shortly previously occurring traumatic, surgical, immunological, medicamentous or toxic previous damaging of the organism (cf. e.g. Bochner, B. S. et al., N. Engl. J. Med., 1991, 3241 1785). Hitherto, a uniform and safe therapy process, especially of the post-traumatic, post-operative, toxic or septic shock has not existed (Barron, R. L., Clin. Pharm. 1993, 12, 829). Dependable criteria for the assessment of the prognosis and of the therapy success in the case of shock illnesses do not exist. Therefore, specific, recognised and rationally based therapy concepts have not been developed, presumably because of the absence of prognostic parameters.

[0003] Therefore, the task exists to find a new process for the diagnostic assessment and monitoring, as well as a medicament for the therapy of states of shock in humans.

[0004] Surprisingly, in the case of the analysis of the blood of shock patients, it was found that some of them, shortly after beginning or in the further course of the shock illness, showed to a considerable extent cyclophilines in the serum of their blood. Patients who, at any point of time during the shock illness (between the first and fifteenth day) show cyclophilines in the serum to a clearly measurable extent died with great regularity in spite of the intensive therapeutic efforts of the treating physician. Patients who survived showed, up to healing, at no point of time in measurable extent cyclophilines in the cell-free parts of their blood (serum, plasma, plasma fractions etc.).

[0005] According to today's state of knowledge, all cells of the human and animal organism, but also many bacteria, plant cells etc., contain a special group of cytoplasmic proteins, the cyclophilines (Galat, A., Eur. J. Biochem. 1993, 216, 689). They serve for the regulation of protein folding, protein stability, protein liberation but also the protein formation in connection with the regulation of the function of cells in the normal state or stress, such as e.g. infections, transplant rejections etc. (Galat v. supra). Cyclophilines have been ascertained not only in the cytoplasm but also in the nucleus of eukaryotic cells (Galat v. supra). However, they do not normally occur in the extracellular medium and no important extracellular effects have been ascribed to them.

[0006] The here-reported observation contradicts the state of knowledge insofar as it is here reported for the first time that in connection with the shock illness in the case of patients in which the shock occurrence finally led to death, already long before their death (days to weeks) considerable amounts of cyclophilines were detectable in the blood. Although the most differing mediators have been found in the case of e.g. shock (de Boer, J. P., Immunopharmacology, 1992, 24, 135), cyclophilines have not been brought into conjunction with the shock occurrence.

[0007] The appearance of cyclophilines in the blood plasma or serum of shock patients is thus a pathognomonicly unfavourable sign. By the monitoring of this parameter in the serum, prognostic predictions can be made and the therapy monitored. A decrease of the cyclophiline concentration is to be regarded as a sign of the possible therapy success, an increase as warning signal for an impending fatal result. Furthermore, in the case of experimentally induced shock states, the cyclophiline concentration can be used as measurable variable for the verification of action of new forms of therapy.

[0008] Furthermore, the here-described surprising finding makes obvious that cyclophilines as mediators are themselves negatively active in the shock occurrences.

[0009] Cyclophilines have similarities with cytokines (Bang et al., Experientia, 1993, 49, 533), a group of substances which participate substantially in the defence preparedness of the human and animal organism. Therefore, their unexpected appearance outside of the cell boundaries could provoke a massive faulty regulation or reregulation of the defence preparedness of the human organism which are responsible for the immediate results of states of shock. For the therapy, it is, therefore, necessary to inactivate, antagonise or to remove the cyclophilines occurring in the case of shock patients. For this purpose, in principle, the following possibilities are available:

[0010] a) blockade or antagonisation of the cyclophilines occurring in the blood plasma by blocking or antagonising pharmaceuticals,

[0011] b) blockade or antagonisation of the cyclophilines occurring by biologically active materials, such as specific antibodies, anti-enzymes etc.,

[0012] c) removal and destruction of the cyclophilines occurring in the blood plasma or serum by physical and chemical methods, such as blood washing etc.

[0013] The detection of the cyclophilines in the serum can take place with different methods, e.g. by enzymological methods, radiochemical methods, immunological methods and classical protein biochemical methods.

[0014] The mentioned diagnostic processes are in part known for the detection of the cyclophilines in cells; further processes are available for other indications and can be adapted to the here-described situations. For example, the cyclophiline concentration can be carried out by the measurement of the rotamase activity (peptidyl-proline-cis-trans-isomerase) according to the following description (H. Bang, Dissertation, Halle, 1986, Wissenschaftsbereich Biochemie, Galat, v.supra and Fischer, G. et al., Biomed. Biochim Acta, 1984, 43, 1101):

[0015] Determination of isomerase activities in body fluids:

[0016] the analysis system depends upon the principle of the coupled spectroscopic enzyme test, i.e. the reaction of an auxiliary enzyme (chymotrypsin) is used for the detection of isomerases,

[0017] by means of a protease (chymotrypsin), the conformation-specific reaction of a chromophoric peptide substrate is carried out and monitored spectroscopically,

[0018] in a first, very rapid reaction, the protease selectively cleaves the trans-conformation,

[0019] in a second, slower phase, the cis-conformation changes uncatalysed into the trans-form and can thereafter be cleaved by the protease,

[0020] isomerases, such as cyclophilines, selectively accelerate this cis-trans conversion,

[0021] thus, in cell-free body fluids, isomerases can be analysed via this spectroscopic detection,

[0022] detection limit: nM of isomerase.

[0023] In a similar manner, with the help of the specific antibodies present against cyclophilines, according to the prior art, immunological, radio-chemical and other processes can also be developed.

[0024] In principle, as therapeutic process, a blockade of the cyclophiline with e.g. cyclosporin A, an agent already used in transplantation medicine, rheumatism therapy, asthma therapy and the therapy e.g. of psoriasis, is clinically usable. Its blocking action on cyclophilines can be verified in vitro.

[0025] In the case of the experimental animal (mouse), the giving of cyclosporin A (CaA) can prevent the lethal state of shock initiated by the injection of bacterial lipopolysaccharide (LPS) or TNFa (tumour necrosis factor a). The liver failure was quantified in this experiment by the detection of liver-typical enzymes, such as ALT_(GPT), AST (GOT) and SDH (sorbitol dehydrogenase). The results are given in Table 1. The data were measured 8 hours after the treatment. The CsA was in each case injected 50 mg/kg i.v. 15 and 1 hour before the treatment.

Table 1

[0026] Protection by cyclosporin A against endotoxin- or TNFa-induced septic liver failure in galactosamine-sensitised mice. ALT [U/1] AST [U/1] SDH [U/1] untreated 40 ± 4  70 ± 8  40 ± 5  control LPS (5 μg/kg) 1500 ± 1025 1044 ± 620  544 ± 340 CsA + LPS 86 ± 61 160 ± 83  25 ± 20 TNF (15 μg/kg) 2650 ± 730  2190 ± 920  1345 ± 370  CsA + TNFa 260 ± 134 210 ± 68  71 ± 49

[0027] Results of Clinical Investigations

[0028] In the Figure are shown concentration measurements of cyclophilines in the serum of 6 patients with shock illnesses. It is shown that, in the case of measurement of cyclophilines, in the case of use of the enzymatic detection method (peptidyl-proline-cis-trans-isomerase, PPlase test) they were detectable only in the case of patients who had increasing or increased amounts of cyclophilines in the plasma. Surprisingly, these intracellular proteins occurred to a clear extent already up to one week before the later death. They increase considerably before death. In contradistinction thereto, all three patients who survived the shock illness showed no measurable increase of the cyclophiline concentration in the serum.

[0029] Cyclophiline concentrations of above 50 U/ml appear, therefore, to point to a lethal ending of the shock situation, concentrations of above 20 U/ml to represent a clear warning signal.

[0030] Quantification and Determination Method:

[0031] The PPlase test is a method variable within limits with reference to the molecule structure of the substrate used and the proteolytic sensitivity of the PPlases, with which even PPlase activities can be determined quantitatively in only coarsely prepared biological materials. For human recombinate cyclophiline, the still detectable enzyme concentration which can be detected lies at about 0.5 ng cyclophiline per ml of serum. In rough approximation, about 1 nM cyclophiline corresponds to a concentration statement of 1 U/ml. In connection with what is said above, it follows that an increase of 5 U/ml thus represents a significant increase of the cyclophiline concentration and is clearly differentiable from a value of 0 U/ml.

[0032] Furthermore, in serum from sepsis patients, not only are clearly quantifiable amounts of cyclophilines ascertained (corresponds to positive values) but also modulators of the cyclophilines. In the case of a negative statement (−40 U/ml), it is a question of inhibitors. These values were determined by examination of the influence of sepsis blood on the cyclophilines given externally to the PPlase test.

[0033] Examples for the determination of the isomerase activity

[0034] making available of cell-free serum;

[0035] use of a UV/VIS spectrophotometer with temperable cuvette container (tempering to 10° C.);

[0036] 1 ml hepes buffer are pre-incubated with 1-20 μl of serum for 30 sec., thereafter 200 μl of the chymotrypsin solution are added thereto and immediately started with 5 μl of a substrate solution;

[0037] the reaction is continuously monitored for 3 min at a wavelength of 390 nm;

[0038] the speed constants of the observed reaction of 1st order is determined with the help of an evaluation programme and corresponds to the U/ml used in the graph;

[0039] a measurement with evaluation requires about 10 min in the case of the present process. 

1. Process for the detection of states of shock and for the therapy control of shock patients, characterised in that the concentration of cyclophilines in the blood of the patients is determined with the help of a) enzymological methods, b) immunological detection methods with the help of specific antibody c) peptide-chemical processes, d) radiochemical methods and serves for the evaluation of the degree of seriousness of the shock.
 2. Process according to claim 1, characterised in that there is used a) as enzymological method, the rotamase activity measurement or, b) as immunological detection method, radio-immunoassays, ELISA enzyme linked immuno-sorben assay or c) as peptide-chemical process, the affinity chromatography or d) the binding of radio-active cyclosporin under special physicochemical reaction conditions.
 3. The use of medicaments, biological active materials and physicochemical methods which reduce the concentration of the active cyclophilines in the blood of shock patients, for the therapy of the shock, especially of the septic shock, of the post-operative shock and of the toxic shock.
 4. Use according to claim 3, characterised in that one employs a) cyclosporins or cyclosporin-related, cyclophiline-inactivating active materials, b) pharmaceuticals with inactivating properties binding specifically or non-specifically to cyclophilines, c) biological inactivators of the cyclophilines, anti-bodies, antibody fragments, cyclophiline-destroying enzymes, d) all forms of blood washing insofar as they are suitable to eliminate cyclophilines or cyclophiline-containing blood fractions. 